Well inserts with brittle membranes and method of manufacturing the same

ABSTRACT

Disclosed is a well insert for cell culture and a method of manufacturing same, where the well insert has a membrane support made of a polyolefin material and having an upper end and a lower end, the upper end being adapted to engage a well of a microplate so as to suspend the well insert therein, and a permeable membrane sealed to the membrane for supporting a tissue culture, the permeable membrane being of brittle material and transparent in both air and water.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of application Ser. No. 15/573,708,filed on Nov. 13, 2017, which is the National Phase under 35 U.S.C. §371 of International Application No. PCT/EP2016/060468, filed on May 10,2016, which claims the benefit under 35 U.S.C. § 119(a) to PatentApplication No. PCT/EP2015/060312, filed in Europe on May 11, 2015, allof which are hereby expressly incorporated by reference into the presentapplication.

TECHNICAL FIELD

The present invention relates to the fields of well inserts for use inmicroplates for cell culture. It relates, more particularly, to wellinserts comprising a brittle membrane made of silicon, ceramic (such assilicon nitride), or similar.

STATE OF THE ART

Well inserts for microplates are used for simulating biologicalbarriers, such as lung, skin, the intestines and the blood-brainbarrier, for in-vitro testing of pharmaceuticals, toxins and othersubstances so as to determine their ability to enter into and movearound within the human or animal body. In vitro models consist of asingle layer or multiple layers of cells that are cultured in thelaboratory so as to mimic the properties of biological barriers in thebody.

A well insert is used in combination with a microwell plate comprising anumber of wells made in plastic. The insert is inserted into a well,which it divides in two, a top (apical) compartment and a bottom(basolateral) compartment which communicate via a porous membrane at thebottom of the insert. Cells are added to the apical side of the wellinsert and are cultured on the porous membrane. Cells may also be addedon the basolateral side and are cultured on that side of the porousmembrane, thereby forming complex stacks of layers of different celltypes in co-culture, with the membrane taking over the role of anon-vascularized support, thereby becoming an internal structure of thebarrier model. Typically, the cells will grow to form a watertight layersitting on or around the porous membrane that divides the apical fromthe basolateral compartment, as in the body.

One of the major players in the well insert industry, Corning, producesa variety of such well inserts, which it commercialises under the names“TRANSWELL®”, “SNAPWELL™”.

The basic TRANSWELL® insert is disclosed in EP 0 239 697, and the moreadvanced “SNAPWELL™” insert, with the membrane supported on a detachablering, is disclosed in U.S. Pat. No. 5,139,951. In each of thesearrangements, the membrane is a porous polymer film, attached to therigid structure of the insert by heat sealing or solvent bonding.

Such polymer films, while cheap, are relatively thick (of the order of10 μm), and the pore size ranges typically from 0.4 to 8.0 μm.Furthermore, due to the mechanical and chemical properties of thepolyester, polycarbonate and collagen-coated PTFE used the reduction ofthe film thickness is extremely difficult.

As a result, interest has been shown in using micromachinable materialssuch as silicon, ceramics such as silicon nitride, alumina and so on forthe membranes. Such materials can be micromachined using MEMS andmicroprocessor-fabrication technologies, and the resulting membranes canhave regions which are significantly thinner than polymer membranes, forinstance of the order of less than 1 μm thick.

EP 2 548 943 cites membranes fabricated by first depositing a thin layerof ceramic material, such as Si₃N₄, on a silicon wafer. Pores are thenetched in the Si₃N₄ by photolithography followed by a dry etch. Thesilicon wafer is then etched from the other side to remove the entirethickness of silicon in selected areas, leaving a set of supports forthe transparent porous membrane that remain after removal of thesilicon. The resulting porous membrane comprises a silicon nitridemembrane supported on a silicon frame which gives it suitable mechanicalproperties. Such a membrane is illustrated in FIG. 1 .

Not only are such membranes significantly thinner than the polymermembranes cited above, but the pore sizes, shapes, densities anddistributions in such a membrane can be tuned as desired with local highreproducibility and precision of the porous pattern; the membranes arehighly transparent in both air and water (polycarbonate membranes aretypically translucent) independently of pore size and density, and themembranes are resistant to acids, bases, solvents, high temperatures ande-beam exposure. They are also reusable (reconditionable) after cellculture.

Furthermore, in comparison with polymer membranes they exhibit lowintrinsic fluorescence of the membrane, which is important fordistinguishing and detecting fluorescence effects of the cells or thesubstances being tested, chemical pretreatment to enhance cell cultureis possible—though not necessary—and they promote good cell growth ingeneral and, in particular, the formation of tight layers of epithelialcells while withstanding common sterilization procedures.

EP 2 548 943 further proposes a cell culture insert for use with suchmembranes. This insert comprises a clamping and a sealing arrangementintended to permit easy removal of the membrane for reconditioning.However, the clamping arrangement requires a significant number ofcomponents, and a significant volume of material in its construction.Insertion and removal of the extremely fragile and brittle membrane isdifficult for the technician, increasing the risk of breaking themembranes.

Various other techniques have been proposed in the art for providingcell culture inserts with membranes secured to a culture support orholder.

WO 2013/081651 A1 discloses well inserts wherein the membrane is heatsealed to the holder by partially melting plastic material of theinsert's holder to create a solid bond joint to the membrane. Suchassembly of the membrane to its holder is theoretically appropriate forattaching membranes of various nature and thickness to a plastic holder.However, it is particularly difficult to control uniform formation of athin, homogenous joint of the melted material about the periphery of themembrane. Additionally, the amount of energy required to melt thesupport material may in some instances affect the physical integrity ofthe membrane depending on the material and thickness thereof.

EP 0 308 129 A1 further discloses a well insert comprising an alumina ormetal oxide membrane being overmoulded in the bottom of a substantiallyconical plastic support. Such technique is however not applicable assuch with very thin fragile brittle membranes of silicon nitride of onlya few micrometres or tenths of micrometres.

An object of the present invention is thus to overcome the disadvantagesof known types of well insert identified above, and thus to facilitateuse of modern, ultrathin, brittle membranes in an economic manner.

DISCLOSURE OF THE INVENTION

More precisely, the invention proposes three main embodiments whichprovide solutions to the above-mentioned problems of the prior art bycombining microfabricated component(s) with a polymer based packagingdesign.

A first embodiment relates to a well insert for cell culture, comprisinga membrane support having an upper end and a lower end, said upper endbeing adapted to engage a well of a microplate so as to suspend the wellinsert therein. The membrane support may be constructed of a singlepiece, or may be a multi-piece arrangement such as in the SNAPWELL™mentioned above, which comprises a separate hanger.

The well insert further comprises a permeable membrane for supporting atissue culture, the permeable membrane being attached at said lower endof the membrane support and sealed thereto, the permeable membrane beingof brittle material, i.e. a material which does not exhibit a plasticdeformation regime and thus will fail before it deforms plastically, andpreferably a membrane having a thickness of less than 10 μm, stillpreferably in the range of 0.1 to 10 μm. Examples of such materials aresilicon, silicon nitride, various ceramics, various glasses, and variousglass-ceramics. To be considered as a “permeable membrane”, at least aportion of the membrane must be permeable—it is not necessary that thewhole area of the membrane be permeable.

According to the invention, the membrane support is made of a polymerexhibiting a linear shrinkage of 1-4% in the radial direction of thepermeable membrane, which is overmoulded on to the permeable membrane soas be sealed thereto. Hence, a brittle membrane can be integrated into awell insert, removing any need for lab technicians to assemble thefragile membranes to the well inserts, making their use simpler. Itshould be noted that the fact that overmoulding has been used isdirectly visible in the finished well insert.

Advantageously, the membrane support is directly sealed to the permeablemembrane, obviating any need for separate sealing pieces. Essentially,the material of the well insert can, due to the use of overmoulding,contact the membrane sufficiently intimately so as to provide a sealedjoint.

Advantageously, the membrane support comprises at least one flange incontact with a first side of the membrane, and at least one opposingflange in contact with a second side of the membrane, said second sidebeing opposite said first side. The membrane is thus “pinched” and heldin sandwich by the material of the membrane support. This improvessealing between the membrane and the membrane support.

The at least one opposing flange may be continuous or may be a pluralityof opposing flanges separated by notches, and the said at least oneflange may be continuous or is formed as a plurality of individualflanges.

The membrane may be recessed with respect to an end face of the membranesupport, thereby avoiding contact between the membrane and a surfaceupon which the well insert is placed. Alternatively, a face, i.e. thelower face, of the membrane may be flush with respect to an end face ofthe membrane support.

Advantageously, the membrane support is of polyolefin material or anyother polymer with a suitable shrinkage during moulding. These materialsprovide good sealing without risking damaging the membrane, and providesthe requisite biocompatibility.

This first embodiment also relates to a method of manufacturing a wellinsert as defined above, comprising the following steps:

-   -   providing a permeable membrane of brittle material;    -   providing a source of molten polymer at a temperature T;    -   providing an injection moulding tool comprising a male part and        a female part;    -   positioning the permeable membrane in the injection moulding        tool;    -   closing the injection moulding tool so as to form a cavity in        which the permeable membrane is situated, the cavity being        shaped so as to conform to the shape of the well insert before        solidification of the molten polymer, i.e. of dimensions        calculated such that, when the polymer has cooled, the desired        shape of the well insert is attained;    -   injecting a quantity of molten polymer into the cavity, the        molten polymer flowing around the periphery of the permeable        membrane and intimately contacting the periphery of the        permeable membrane;    -   hardening the molten polymer such that it applies a radial force        around the periphery of the permeable membrane;    -   opening the moulding tool;    -   removing the well insert from the moulding tool.

This method provides an economic method for producing well insertscomprising a brittle membrane, which obviate the need for a technicianto handle the membranes directly.

Advantageously, the material of the molten polymer, the shape of thecavity, and the initial temperature T of the molten polymer are chosensuch that, upon cooling to room temperature and solidifying, the polymerexhibits a linear shrinkage of 1-4%, preferably 1.5-2.5% in the radialdirection of the permeable membrane. This provides good sealing, whilereducing the risk of breaking the membrane during moulding or duringhandling of the well insert. The skilled person knows how to perform therequired calculations for a given size and shape of well insert.

Advantageously, the permeable membrane is positioned in the injectionmoulding tool by means of a vacuum.

Advantageously, the male part of the injection moulding tool comprises aseat shaped to receive a membrane. The male part of the injectionmoulding tool may further comprise at least three abutments distributedaround said seat, said abutments being adapted to position the permeablemembrane radially. Good support of the

membrane in the mould is thus obtained, reducing risk of applyingundesired bending or torsion to the membrane during injection mouldingand thereby reducing the risk of breaking the membrane.

Advantageously, the abutments have a height of at least 0.75 mm and nomore than 20% of the thickness of the permeable membrane. The abutmentsare thus sufficiently large to tolerate a certain degree of wear, andyet are not so large as to cause weak spots in the contact between themembrane and the membrane support. Such weak spots may be inadequatelysealed, and hence their avoidance is desirable.

The male part of the injection moulding tool may also further compriseat least one abutment shaped so as to fit into a hollow surface featureof the membrane. This provides another way of supporting and positioningthe membrane in the mould which eliminates the need for abutmentssituated on the outside of the membrane.

The abutments, whatever their configuration, may be provided on aremovable insert. They can thus be easily replaced if they are worn outwithout having to replace the entire mould.

Advantageously, the female part of the injection moulding tool comprisesa vent to permit escape of air during injection of the polymer material,the vent being axial with respect to the membrane. Since this vent isaxial with respect to the membrane, it allows air inside the mould to bedisplaced evenly, allowing the polymer melt to flow evenly through themould and around the membrane, improving the quality of the moulding andpreventing uneven application of force by the melt to the membrane.

Advantageously, when the injection moulding tool is closed, a play of2-4 μm is present between a flat surface of the permeable membrane and asurface of the female part of the injection moulding tool which facessaid flat surface. This play is sufficient to permit air to escape,however is not so great as to permit the membrane to tilt so much thatthe polymer melt may flow under one side and apply excessive bending ortwisting forces thereto that could cause the membrane to break

A second embodiment of the invention relates to a well insert for cellculture, comprising a membrane support having an upper end and a lowerend, said upper end being adapted to engage a well of a microplate so asto suspend the well insert therein. As for the first embodiment,membrane support may be constructed of a single piece, or may be amulti-piece arrangement such as in the SNAPWELL™ mentioned above, whichcomprises a separate hanger.

The well insert further comprises a permeable membrane for supporting atissue culture, the permeable membrane being attached at said lower endof the membrane support and sealed thereto, the permeable membrane beingof brittle material, i.e. a material which does not exhibit a plasticdeformation regime, and comprising surface features arranged in asurface thereof. Examples of such materials are silicon, siliconnitride, various ceramics, various glasses, and various glass-ceramics.To be considered as a “permeable membrane”, at least a portion of themembrane must be permeable—it is not necessary that the whole area ofthe membrane be permeable.

According to the invention, the membrane support is fastened to thepermeable membrane in the surface features thereof.

In the context of the present invention, the term “fastened” should beunderstood under its standard meaning, i.e. attached firmly andsecurely, especially by pinning, tying or nailing. The fastening of themembrane support to the membrane is achieved according to the inventionby insertion and retention of the membrane support into said surfacefeatures arranged in the membrane. Said insertion of the membranesupport is advantageously achieved by melting the membrane supportmaterial and/or a surface layer of the membrane at their interface, e.g.by laser, infrared heating, thermal contact with a hot plate, orapplication of ultrasonic energy, so as to allow molten material to wetsaid interface and to flow into the surface features, which are arrangedin the membrane surface at the periphery thereof and by subsequentcooling of the molten material, either at ambient temperature or byexternal cooling. Upon cooling the molten material actually bonds thematerial support and membrane uniformly and homogeneously about themembrane periphery and further mechanically fastens said support throughformation of hardened protrusions forming pillars or nails in themicrostructures. Such fastening improve the strength and sealing of theconnection between the membrane support and the membrane

An alternative solution to overmoulding is thus proposed for adirectly-integrated well insert, which comprises the same advantages asgiven above in relation to the first embodiment.

Advantageously, the membrane comprises a joining zone on a planarsurface thereof, said planar surface facing said membrane support andcomprising said surface features into and/or around which the materialof the membrane support extends.

The surface features may comprise at least one of protrusions (such asridges and lugs), recesses (such as grooves and notches), and undercutportions. Particularly interesting are grooves provided with undercuts,which may be produced by anisotropic etching along the crystal planes ofthe material of the membrane. Such surface features may be combined,e.g. by using at least one protrusion and at least one recess, said atleast one recess ideally being undercut. Each of said protrusion andsaid recess may extend around the periphery of the membrane, optimizingsealing.

Advantageously, said protrusion is situated towards the periphery of themembrane.

Advantageously, the surface features comprise at least one recess formedas a first groove, said first groove extending around the membrane on aplanar surface thereof, said surface features further comprising aplurality of further grooves arranged perpendicular to said firstgroove. This arrangement provides excellent fastening, actuallyanchoring and sealing.

This second embodiment further relates to a method of manufacturing awell insert as defined above, comprising the steps of:

-   -   providing a permeable membrane of brittle material comprising        surface features in a surface thereof;    -   providing a membrane support made of plastics material;    -   placing said membrane support in contact with a planar surface        of said membrane;    -   melting said plastics material in contact with said membrane so        as to allow the molten material to flow into the surface        features, and    -   allowing the molten material to cool so as to fasten said        membrane support to said membrane.

This method thus results in the production of the well insert as above,which has the noted advantages.

Advantageously, said step of melting said plastics material comprisesheating by means of at least one of:

-   -   laser energy, either through the membrane or from the side;    -   ultrasonic energy;    -   thermal contact with a hotplate    -   infrared radiation.

Advantagously, the surface features may comprise at least one recess orgroove, and said step of melting said plastics material may comprisecausing said plastics material to flow into said recess or groove. Thisprovides good sealing and good anchoring upon cooling of the moltenplastics material.

The third embodiment of the invention relates to a well insert for cellculture, comprising: a membrane support comprising an hanger having anupper end and a lower end, said upper end being adapted to engage a wellof a microplate so as to suspend the well insert therein, and apermeable membrane for supporting a tissue culture, the permeablemembrane being in arranged at said lower end of the hanger and sealedthereto, the permeable membrane being of brittle material. This membraneis as defined above in reference to the first and second embodiments.

According to the invention, the membrane support comprises a sealarranged at said lower end of said hanger, which may be integral withthe hanger or may be a separate piece, and the membrane support furthercomprises an end piece releasably clipped to said hanger so as tosupport said membrane between said end piece and said hanger in contactwith said seal. In other words, the membrane support comprises twopieces. The end piece is arranged so as to cause said membrane tocompress said seal upon clipping of the end piece to said hanger.

Thus, a simpler, more compact arrangement to that described in EP 2 548943 is provided. This arrangement requires less material to produce, andpermits easier handling of the membrane since it no longer needs to beplaced at the bottom of a bore.

The end piece is furthermore thus situated outside of said hanger.

Advantageously, the end piece comprises a plurality of arms extendingtowards said upper end and comprising firstclipping elements eachinterfacing with a corresponding second complementary clipping elementsprovided on said hanger. Such an arrangement facilitates “clipping” theend piece onto the hanger so as to compress the seal. These firstclipping elements may be hooks, and/or the second clipping elements maybe openings provided in said hanger. Alternative arrangements are alsopossible.

BRIEF DESCRIPTION OF THE DRAWINGS

Further details of the invention will appear more clearly upon readingthe following description, in reference to the annexed drawings, whichshow:

FIG. 1 —a typical permeable membrane made of a brittle material;

FIG. 2 —a perspective view from underneath a well insert according to afirst embodiment of the invention;

FIG. 3 —a cutaway view of the lower end of the well insert of FIG. 2 ;

FIGS. 4 and 5 —perspective views of a male part of an injection mouldingtool for producing a well insert as shown in FIG. 2 ;

FIGS. 6, 7 and 8 —detail views of elements of male and female parts ofthe injection moulding tool in relation to a membrane placed therein;

FIG. 9 —a variant of a male part of an injection moulding tool;

FIG. 10 —an insert for the male part of an injection moulding tool ofFIG. 9 ;

FIGS. 11 and 12 —views similar to FIGS. 9 and 10 respectively showing avariant of a male part of an injection moulding tool;

FIGS. 13, 14 and 15 —cutaway views of further variants of well insertsaccording to the first embodiment in which the lower surface of themembrane is flush with the lower surface of the membrane support;

FIG. 16 —a cutaway view of a female part of an injection moulding toolfor producing a well insert as illustrated in FIG. 13 or 14 ;

FIG. 17 a —a membrane specifically adapted for producing a well insertaccording to the second embodiment;

FIG. 17 b —a cutaway view of the membrane of 17 a joined to a membranesupport;

FIG. 18 a-j —schematic views of various arrangements of structuralfeatures for improving the attachment between the membrane and themembrane support; and

FIGS. 19 and 20 —photographs and a cross-sectional schematic view of awell insert according to the third embodiment of the invention.

EMBODIMENTS OF THE INVENTION Generalities

In the figures, the same reference signs have been used throughout toindicate the same or equivalent parts.

FIG. 1 illustrates an example of a membrane 1 typical for use with wellinserts according to the invention. Although only a portion of membrane1 is actually porous, for ease of reading the terms “membrane” and“permeable membrane” are used interchangeably to apply to the entirepiece 1, which is appropriate since it comprises porous sections and isthus at least partially porous. Membrane 1 is typically made of silicon,silicon nitride, silica or other brittle material (e.g. ceramics,glasses, glass-ceramics) without a plastic deformation regime, i.e. willbreak without previously being permanently deformed. Membrane 1comprises a thicker portion section 1 a around the periphery of themembrane 1, and a plurality of thinner portions 1 b, which arestructured with pores so to be permeable to e.g. ions, water, cellculture media, biological molecules and so on as according torequirements. Thinner portions may be structured by any convenientprocess such as machining, grinding, laser etching, wet etching,reactive ion etching, anisotropic etching of microstructures, and so on,to create a desired porosity. Between individual thinner portions is agrid structure of thicker material 1 c, which define recesses at thebottom of which are situated the thinner portions 1 b. Frame section 1 cprovides structural integrity to the membrane 1. Thicker portion 1 a andthe grid structure 1 c are typically of the order of 200-1000 μm thick,whereas the thinner portions 1 b are typically of the order of 100 nm to10 μm thick, this latter thickness normally being chosen in function ofthe desired pore diameters, and are as a result very fragile. Indeed,the overall membrane is exceedingly fragile and thus difficult to handlein both a manufacturing setting and in the laboratory. Themicrofabrication techniques used to create the membranes and pores maybe also used to further functionalize either side or both sides of themembrane, such as depositing another thin layer of metal or dielectricto reduce pore diameter or exploit non conventional effect such as lightsurface interaction (plasmonics), or by adding electrodes,thermocouples, local heaters or coolers, sensors or sensor arrays fortemperature, pressure, photodetectors, actuators, MEMS, microfluidicsystems, etc., local processing power to condition and read/write outdata generated by any electronic components formed thereupon. Differenttypes of functionalization can be identified e.g. by colour coding thewell insert 10.

It is upon the surface of the membrane, and particularly in contact withthe thinner portions 1 b, that cells will be cultured.

For ease of reading, the face of the membrane 1 intended to face theoutside of the well insert 10 is considered to be the “lower” face, andthe face of the membrane 1 intended to face the inside of the wellinsert 10 is considered to be the “upper” face.

Likewise, the end of the well insert 10 closed by the membrane 1 isconsidered to be the “lower” end, and the open end of the well insert 10is considered to be the “upper” end.

Embodiment 1: Overmoulding

FIG. 2 illustrates a perspective view from below of a well insert 10according to a first embodiment of the invention.

Globally, the structure of the well insert 10 is similar to theTRANSWELL® insert mentioned in the introduction, and comprises amembrane support 10 a supporting membrane 1. As such, at a first, openend it comprises a flange 11 sized to interface with a well of amicroplate (not illustrated) so as to suspend the well insert 10therein. At a second end 12, the well insert 10 supports the membrane 1as will be described below. Adjacent to the second end 12 is a firstintermediate section comprising an impermeable sidewall 13 which,together with the membrane 1, form a vessel constituting an apicalcavity when suspended in the well of a microplate. Joining theimpermeable wall to the flange 11 is a second intermediate section,comprising a plurality of openings 14 divided by connecting elements 15.These openings permit access to the basolateral compartment of the wellof the microplate when in use.

Membrane support 10 a is constructed of plastics material, as isgenerally known. For reasons that will be given below, polyolefinmaterial is particularly suitable, however other plastics are certainlypossible.

FIG. 3 illustrates a perspective sectional view of the well insert ofFIG. 2 , focussing on the second, lower, end 12.

The membrane 1 is integrally moulded into the structure of the secondend 12 of the well insert 10. Up to now, this has not been successfullyachieved. The types of materials mentioned above for the membrane 1 areexceedingly fragile in bending, torsion and shear. They have excellentmechanical strength in compression, however in view of the forcesrequired to ensure hermetic sealing directly between the material of themembrane support 10 a and the membrane 1, if there is any asymmetry inthe distribution of forces, torsion or bending of the membrane 1 willoccur. Indeed, early injection moulding experiments resulted in thecomplete destruction of the membrane 1, it having been reduced to powderduring the injection moulding process. It is largely for this reasonthat it was not an obvious choice to use injection moulding, and indeedsignificant effort was required to successfully achieve overmoulding.

As can be seen in FIG. 3 , in the finished well insert 10, membrane 1 issupported from underneath (i.e. on the exterior side of the well insert10) by an annular flange 16, extending around the periphery of themembrane 1 and towards the centre of the membrane 1 for a certaindistance. On the opposite side of the membrane 1, i.e. on the upper side(on the inside of the well insert), the membrane 1 is supported by aplurality of opposing, arcuate, flanges 17 separated by notches 18. Inthe example illustrated, three opposing flanges 17 are separated bythree notches 18 distributed at equal angular intervals around themembrane 1. Other numbers are likewise possible, however three appearsto be optimal. The peripheral sidewall of the membrane 1 is also incontact with a corresponding cylindrical wall 12 a of lower end 12 ofthe membrane support 10 a. The significance of these notches will becomemore apparent below in the discussion of the corresponding injectionmoulding tool. It should be noted that at each of the notches 18,material of the membrane support 10 a is not present on the upper sideof membrane 1.

Due to the presence of annular flange 16, the membrane 1 is recessedfrom the lower surface of the membrane support 10 a and thus cannot notcome into contact with a surface on which the well insert 10 is placed.

Experiments have shown that the choice of material, particularly but notexclusively polyolefin material, the temperature of the melt, thedimensions of the flanges 16, 17 and the thickness of the materialadjacent to the cylindrical wall 1 e of the membrane 1 are chosen so asto exhibit, in the theoretical absence of the membrane 1, a 1-4%, bettera 1.5-2.5%, even better a 2% linear shrinkage. In the case ofpolyolefin, PMMA, or polycarbonate, a melt temperature of approximately250° C. achieves this shrinkage. In the case of PEEK, approximately 420°C. is more appropriate. Other polymer materials and other melttemperatures are of course possible. As a result, a force is appliedradially on the cylindrical wall 1 e of the membrane 1, as well as anaxial clamping force applied between flanges 16 and 17, is sufficient tohermetically seal the membrane 1 to the membrane support 10 a, while theforces are insufficient to damage the membrane 1 either during mouldingor in use.

FIGS. 4 and 5 illustrate a male part 30 of an injection moulding tooladapted to form a well insert 10 according to the invention, and FIG. 6illustrates a cutaway view of the male part 30 with a membrane 1 placedthereupon, the cut passing diametrically through an abutment. Themajority of the features of the mould, such as the protrusions forforming openings 14, are standard and need not be described further.Male part 30 of the mould protrudes from a face 31 of the mould, as isgenerally known. Face 31 of the mould comprises a deep melt distributiongroove 32 and with three melt outlets 33 leading from the meltdistribution groove 32 to the cavity formed when the mould is closed andwhich will become the membrane support 10 a once moulded. Other numbersof melt outlets are of course also possible. Melt outlets 33 are evenlydistributed around the mould, and ideally are of the same number as theabutments 34 (see below) which will form notches 18 in the finished wellinsert 10. Furthermore, each melt outlet 33 is ideally aligned with acorresponding abutment 34 to minimise the formation of voids adjacent tothe abutments 34, although it is possible to arrange the melt outletsdifferently. However, arranging them in line with each other causes themelt to flow evenly and symmetrically around each side of each abutment34, and thus minimises any unbalanced forces applied to the membrane 1by the flowing of the melt.

Abutments 34 are provided adjacent to a support surface 35, upon whichthe membrane 1 is positioned for injection moulding. These abutments 34are situated radially outboard of the support surface 35, and extendperpendicularly to the support surface 35 in such a manner as to contactthe cylindrical sidewall 1 p of a membrane 1 placed upon the supportsurface 35. In the illustration of FIGS. 4 and 5 , support surface 35 isannular and extends around the free end of the male part 30 of theinjection moulding tool, as well as radially towards each abutment 34.Aside from around each abutment 34, the membrane 1 protrudes outwardsfrom support surface 35, as can be more clearly seen in FIG. 6 . Thelateral surface 34 a of each abutment 34 is curved so as to closelyfollow the outer surface 12 a of the membrane 1, with sufficient play toenable the membrane 1 to be placed therein easily without damaging it. Aplay of 15-35 μm, preferably 20-30 μm, ideally substantially 25 μm issufficient.

This play 36 is visible on FIG. 7 , between abutment 34 and membrane 1.Also visible on FIG. 7 is the height of abutment 34 over the supportsurface 35. This height should be kept to a minimum so as to ensuremaximum contact around the periphery 1 p of the membrane 1 with theplastics material of the membrane support 10 a. However, the material ofthe membrane is significantly harder than the metal material of the malepart of the mould 30, and hence wear of the abutments 34 is an issue, asis their machinability. In practical terms, a height of 0.075-0.2 mm isadequate. In terms of sealing, a relative height between the abutments34 and the thickness of the membrane 1 of 1:20 to 1:5, ideally 1:10 isagain adequate.

Inside of support surface 35 is relieved so as to provide a recess 37.In order to position and gently hold the membrane 1 while the injectionmoulding tool is being closed, a vacuum line (not illustrated) may beprovided opening into recess 37, however this is not obligatory.

FIG. 8 illustrates a cutaway view of a complete and closed injectionmoulding tool, with a membrane 1 placed therein. This view is in theopposite orientation to that of FIGS. 5-7 . The entire tool comprisesnot only male portion 30 as described above, but also female portion 40.

When the male 30 and female 40 part of the injection moulding tool areclosed on the membrane 1, an axial play 38 is present between themembrane 1 and the female 40 or male 30 part of the tool, depending onthe orientation of the tool and whether a vacuum is present or not. Thisplay 38 is ideally between 1 and 5 μm, preferably between 1.5 and 2.5μm.

The play 38 has several functions. Firstly, it prevents the tool, whenclosed, from crushing the membrane 1. Secondly, it permits air to escapethrough a vent 42 provided for this purpose in the female part 40 of theinjection moulding tool. Additionally or alternatively, such a vent canbe provided in the male part 30 of the tool. Since, as is clear from thefigures, the cavity formed between the two parts 30, 40 of the injectionmoulding tool is filled from the wider end, i.e. the end that forms theflange 11 of the well insert 10, air will be displaced and must exitfrom the membrane 1 end of the cavity in an even fashion to prevent airbubbles forming. Air bubbles are not only undesirable manufacturingflaws, but they can result in differential forces being applied to themembrane 1 due to irregular melt flow, potentially twisting or bendingmembrane 1 and thus destroying it.

It should further be noted that there also exists a risk of damaging thewell insert 10 due to careless handling while extracting it from themould. To minimise this risk, the sidewall 13 of the first intermediatesection is provided with a taper of 7-8°. This range has proved optimalfor this application.

Since membrane 1 is not only brittle but is very hard, and certainlyharder than the material of the injection moulding tool, abutments 34can be subject to wear due to the membrane 1 rubbing there against whenit is positioned on the male part 30 of the injection moulding tool, andduring injection of the plastics material.

FIGS. 9 and 10 illustrate a modification to the male part 30 of theinjection moulding tool which permits replacement of the abutments 34without requiring reworking of the entire tool.

As can be seen in FIG. 9 , the male part 30 the injection moulding toolcomprises a bore 39, and terminates in an end surface 43. Asillustrated, and surface 43 is flat, however it may also be convex,concave, undulating, crenellated or any other convenient shape. Andsurface 43 may be provided with positioning means such as pins, lugs,crenelations or similar so as to angularly position an insert 44.

Insert 44 is provided with a stem 45 sized to extend down bore 39, stem45 being coaxial with insert head 46 which carries abutments 34, supportsurface 35 and recess 37 as defined above.

Insert 44 may also be positioned on the male part 30 of the injectionmoulding tool e.g. by pinning, threading, or any other convenientpositioning means.

Thus, in the case of excessive wear of abutments 34, insert 44 cansimply be replaced.

FIGS. 11 and 12 illustrate an alternative arrangement of insert 44. Inthis variant, male part 30 of the injection moulding tool retain supportsurface 35, and abutments 34 are formed directly on insert 44 in theform of lugs shaped so as to fit closely inside the recesses formed byframe elements 1 c and thinner portions 1 b of the membrane 1, asillustrated in FIG. 1 . In FIG. 12 , membrane 1 has been cut along itsdiameter so as to illustrate this principle.

In such case, insert 44 may be made of the same or similar material tothe rest of the male part 30 of the injection moulding tool, or may bemade of a softer material such as a plastic. It is also conceivable thatinsert 44 may be made of a harder material such as a ceramic which issufficiently hard so as not to be subject to wear from contact with themembrane 1. For instance, a sintered ceramic material would be suitable,and may be bonded into bore 39 with a suitable adhesive.

When using such an insert, no notches 18 are present in the finishedwell insert is due to the absence of abutments 34 situated outside ofthe support surface 35. In such a case, opposing flange 17 extends alongthe entirety of the periphery of the membrane 1 and is thus annular.

For cases in which a recessed membrane is undesirable, it is alsopossible to arrange the lower surface of the membrane 1 to be flush withthe lower surface of membrane support 10 a. FIGS. 13 and 14 illustrate 2variants of such an arrangement.

In FIG. 13 , membrane 1 is provided with an annular rim 1 r arrangedextending around an upper side thereof. Annular flange 16 of themembrane support 10 a and thus extends around the periphery of membrane1 up to the full-thickness section so as to hold the membrane 1 betweenannular flange 16 and one or more opposing flanges 17 situated on theupper side of membrane 1, the number of opposing flanges depending onwhich of the above-mentioned arrangements of abutments 34 is used.

In FIG. 14 , membrane 1 is provided with a plurality of peripheralnotches 1 n spaced at substantially equal annular intervals around theperiphery on the lower face of the membrane 1. Once moulded, a pluralityof flanges 16 are formed extending into the corresponding peripheralnotches 1 n, thereby supporting the membrane 1 between these flanges 16and one or more opposing flanges 17 as above.

For the membranes 1 illustrated in FIGS. 13 and 14 , annular rim 1 r andperipheral notches 1 n may be formed e.g. by etching, laser machining,or grinding.

FIG. 15 illustrates yet further flush-membrane variant, adapted for asquare membrane 1. Square membranes 1 are interesting from amanufacturing perspective, since many more of them can be fitted onto asingle wafer of silicon, a silicon nitride or similar, and they can veryeasily be separated therefrom simply by cutting the wafer in straightlines.

In the variant of FIG. 15 , the membrane 1 is provided with a peripheralrim 1 r, in a similar fashion to FIG. 13 , and no notches 18 are presentin the membrane holder 10 a. Flanges 16 and opposing flanges 17 are thusrectilinear, following the periphery of membrane 1.

Alternatively, peripheral notches 1 n as in FIG. 14 may be used, andmembrane 1 may be positioned on the male part 30 of the injectionmoulding tool by abutments 34 similar to those illustrated in FIGS. 4-7and 10 but numbering four and being situated either at the midpoint ofthe sides of membrane 1 or at the corners of membrane 1, therebyresulting in corresponding notches 18 being present in the finished wellinsert 10.

In order to create such a flush-fitted membrane, particular adaptationof the female part 40 of the injection moulding tool is required toprevent the lower face of the well insert 10 from being convex, with themembrane 1 protruding outwards. This is caused by the shrinkage of thematerial back towards the upper end of the well insert 10 during coolingof the plastics material during moulding. Such protrusion of themembrane 1 is not only undesirable from an aesthetic perspective, but itcan also reduce the quality of the sealing between the membrane 1 andthe membrane holder 10 a, as the flange 16 is pulled backwards andoutwards, and the cylindrical wall 12 a of plastics material whichshould be in intimate contact with the peripheral wall le of themembrane 1 is likewise pulled backwards and outwards.

To solve this problem, the female part 40 of the injection moulded toolis formed as illustrated in FIG. 16 . In essence, the cavity 40 a in thefemale part 40 surrounding the membrane 1 extends away from the membrane1 (illustrated here for reference) such that, before the plasticsmaterial cools, membrane 1 is recessed from the end face of well insert10. The extent to which the cavity 40 a extends ahead of the membrane 1is calculated such that, upon cooling of the plastics material to roomtemperature, the lower end face of the well insert 10 is absolutelyflush and flat. This calculation depends on the properties of theplastics material chosen, the temperature of the melt, and the shape ofthe well insert 10. Based on these parameters, the person skilled in theart can make the required calculations.

It should be further noted that the same principle applies to atwo-piece well insert of the SNAPWELL™-type. In such a case, themembrane 1 is integrally moulded to a first element of the membranesupport 10 a which attaches to a second element constituting a separatehanger so as to form the entire well insert, in the manner known for theSNAPWELL™.

Embodiment 2: Direct Fastening

FIGS. 17 a-18 h illustrate an alternative principle for joining themembrane 1 to the membrane support 10 a so as to form a well insertaccording to the invention.

FIG. 17 a illustrates, in a view similar to FIG. 1 , a membrane 1adapted for fastening to a membrane support 10 a by melting saidmembrane support partially for it to fill surface features, inparticular microstructures, arranged in the membrane to provide pinningor nailing of the support to the membrane and solid attachmenttherebetween upon cooling of the molten material without any externallinking element. While melting is generally known for attaching plasticparts to hard part is, e.g. ceramics, glasses, glass-ceramics and so on,to the best of the applicants knowledge it has never been applied forconstruction of a well insert 10.

Examples of such joining are given in the documents EP2061589,EP2735432, and US2011232826. In essence, such methods permit joining aplastic part to a hard part without use of any third-party mediatorssuch as glue, solder, intermediate metallic layers, or similar, bybringing two parts into contact and then causing the material of one ofthe parts to melt and thereby weld itself to the other part.

Membrane 1 is similar to that of FIG. 1 , except that it comprises ajoining zone 50 situated at or near its periphery on the upper surfacethereof. Joining zone 50 is structured by any convenient process such asmachining, grinding, laser etching, wet etching, reactive ion etching,anisotropic etching of microstructures, and so on. Examples of suchanisotropically etched microstructures particularly suited for wellinserts will be described in more detail below. Although the membrane 1has been illustrated as being circular, a square membrane as illustratedin FIG. 15 is particularly suitable in the case of anisotropicallyetched structuring, due to straight lines being easier to etch thancurves.

FIG. 17 b illustrates, in a partial, transparent cutaway view, aschematic view of membrane 1 of FIG. 16 joined to sidewall 13 of the 1^(st) intermediate section of membrane support 10 a. The remainder ofthe membrane support 10 may be constructed as that illustrated for the 1^(st) embodiment, and has not been illustrated here. Furthermore, eventhough sidewall 13 has been illustrated here as being cylindrical, itmay also be tapered as in the 1 ^(st) embodiment.

Sidewall 13 is joined to the joining zone 50 of membrane 1 by directlythermally welding the sidewall 13 to the joining zone 50. This can beachieved, as is generally known, by heating, e.g. by means of a laserdirected through the membrane or from the side, ultrasonic vibrations,or direct application of heat, which causes the lower end of sidewall 13to soften and adhere to the structure and texture of joining zone 50. Toassist in this joining, the lower end of sidewall 13 may be providedequally with structures such as illustrated in EP2735432, referencedabove. Suitable materials for the membrane support 10 a embodiment arepolystyrene and polycarbonate, although of course other plasticsmaterials such as polyolefins are also possible.

FIGS. 18 a-j illustrate variants of structuring joining zone 50, incross-section. These variants apply equally well to both circular andsquare/rectangular membranes 1. In the case of circular membranes, thefeatures illustrated can be presumed to be cylindrically symmetrical, orsymmetrical with a certain order of symmetry (e.g. greater than twelve)if the features are formed as facets rather than as smooth curves aroundthe periphery of the membrane. If a feature is microfabricated with ananisotropical method, the anisotropy will impose the orientation of thefeature locally, e.g. the crystal structure of silicon may favour foursymmetrical orientations of a feature, which does not impede its globalarrangement with higher symmetry.

FIG. 18 a shows a single protruding ridge 51 of rectangularcross-section, and FIG. 18 g shows a single cavity 51 a, both of whichcan e.g. formed by isotropic etching (e.g. reactive ion etching (RIE),deep reactive ion etching (DRIE) or similar) using a mask of e.g.silicon nitride which is then subsequently removed. Ridge 51 or cavity51 a assist in bonding by providing increased surface area, and alsoimproved sealing. Although ridge 51 and cavity 51 a have beenillustrated as being off-centre towards the inside of the well insert10, they may be situated on the centreline of sidewall 13, or evenoff-centre towards the outside of the well insert 10. Ridge 51/cavity 51a extend around the entire periphery of the membrane 1, and may beinterrupted to provide improved adhesion or improved sealing.

FIG. 18 b shows an embodiment with a pair of protruding ridges 51,further improving sealing. Naturally, this also applies to cavities 51a, or to a mixture of ridges 51 and cavities 51 a

FIG. 18 c illustrates an embodiment in which the protruding ridge 51 isof trapezoidal cross-section, and FIG. 18 h shows a cavity 51 csimilarly of trapezoidal cross-section, formed e.g. via anisotropicetching (plasma etching, wet etching, or similar) using a mask 52 ofe.g. SiN or SiO₂. In the example of FIG. 18 c , the mask 52 has not beenremoved.

FIG. 18 d illustrates an embodiment in which, instead of a protrudingridge, and undercut “lock” structure 53 is provided, again extendingaround the entire periphery of membrane 1. Formation of such structuresby isotropic etching of a straight-sided cavity by means of a mask ande.g. RIE or DRIE followed by anisotropic etching by e.g. plasma etchingor wet etching along the <111>crystal planes is generally known and neednot be discussed further. FIG. 18 e illustrates a combination of anundercut “lock” structure 53 towards the inside, and a protrusion 51 tothe outside of the joining zone 50. Naturally, the structures may beprovided in the opposite arrangement.

FIG. 18 f illustrates a variant in which two undercut “lock” structures53 are provided adjacent to one another, so as to form a ridge 56between them. This ridge 56 forms an excellent seal. As illustrated, thematerial of the sidewall 13 has been melted to a greater extent than inthe preceding variants, so as to form radiuses 54 on either side of thesidewall 13. Excess material 55 can flow into the outermost “lock”structure 53. A third “lock” structure may also be provided on theinside of the well insert 10, i.e. to the left of the sidewall 13 in theillustration of FIG. 18 f.

FIG. 18 i illustrates just the joining zone 50 of a membrane 1, which isprovided with a more complex etched structure. This etching is carriedout by means of appropriate masks so as to create a series ofinterconnecting “lock” structures 53 leaving ridges 56 a, 56 btherebetween. Ridge 56 a is similar to ridge 56 of FIG. 18 f , andextends around the periphery of the membrane 1 on the outer side ofridges 56 b. Ridge 56 a serves as the primary seal. Ridges 56 b aremutually parallel, and are arranged perpendicular to ridge 56 a, andserve primarily for anchoring the sidewall 13 to the membrane 1.

Finally, FIG. 18 j illustrates a variant in which interconnecting “lock”structures 53 are formed in a grid pattern so as to leave an array ofprotuberances 56 c.

Third Embodiment: Clipping

FIGS. 19 and 20 , illustrate a third embodiment of a well insert 10according to the invention, the left-hand image of FIG. 19 showing wellinsert 10 in disassembled form, and FIG. 20 showing schematically asection along A-A of FIG. 19 .

Well insert 10 according to this embodiment comprises a membrane support10 a formed in two parts, namely hanger 70 and end piece 60. Hanger 70is constructed in a similar fashion to the entire membrane support 10 aas shown in FIGS. 2, 13 and 14 , except that it does not integrallycomprise the membrane 1. However, other features such as sidewall 13,openings 14, connecting elements 15, flange 11 etc. remain substantiallysimilar. Indeed, hanger 70 may also be a Corning SNAPWELL™ hanger, orany other commercially available hanger.

Membrane 1, which is of the type as described above, is positionedagainst the lower end of sidewall 13 by means of an end piece 60, and issealed thereto by means of a seal 65. As illustrated, seal 65 is aseparate piece arranged in an annular groove 65 a extending around thelower end face of hanger 70, although it may be a seal integral with thesidewall 13, e.g. by being formed of a sufficiently soft elastomericmaterial co-moulded with the sidewall 13. Seal 65 may also be a simpleflat seal, which does not require an annular groove 65.

The membrane 1 is held in sandwich between a flange 61 of the end piece60, which extends inwards leaving an opening 62 to permit fluid accessto the surface of the membrane 1. End piece 60 is thus situated on theoutside of hanger 70.

End piece 60 comprises a plurality of arms 63 extending towards openings14, which terminate in first clipping elements 64 such as hooks or lugs,adapted to interface releasably with the lower edges of correspondingopenings 14, which constitute corresponding second clipping elements.Alternatively, the second clipping elements may comprise one or morelugs, rims, recesses or other features may be provided extending into orout from the structure of hanger 70, which are shaped so as to interfacewith corresponding first clipping elements provided on arms 63. Asillustrated, these first clipping elements 64 are hooks engaging withthe openings 14.

The length of the arms 63 and the position of the clipping elements 64are chosen so as to, in the assembled state, compress seal 65. This notonly seals the periphery of membrane 1 to the hanger 70, but also servesto keep the arms 70 in tension and the attachment means 64 engaged. Itis also possible that arms 70 provide an elastic force in addition.

In essence, end piece 60 thus clips onto hanger 70 so as to maintain themembrane 1 in place, while permitting easy removal thereof by unclippingthe end piece 60 from the hanger 70.

This permits the well insert 10 to be disassembled for cleaning themembrane 1 for re-use.

Although the invention has been described in reference to variousconcrete embodiments as described above, these are not to be consideredas being limiting to the scope of the invention. Further variants arepossible without departing from the scope of the invention as defined inthe appended claims.

1. A well insert (10) for cell culture, comprising: a membrane support(10 a) made of a polyolefin material and having an upper end and a lowerend, said upper end being adapted to engage a well of a microplate so asto suspend the well insert (10) therein; and a permeable membrane (1),transparent in both air and water, for supporting a tissue culture, thepermeable membrane (1) being attached at said lower end of the membranesupport (10 a) and sealed thereto, the permeable membrane (1) being ofbrittle material and comprising surface features arranged in a surfacethereof, wherein the membrane support (10 a) is fastened to thepermeable membrane (1) in the surface features in the surface thereof.2. The well insert (10) according to claim 1, wherein the permeablemembrane (1) comprises a joining zone (50) on a planar surface thereof,said planar surface facing said membrane support (10 a) and comprisingsaid surface features (51, 52, 53, 56, 56 a, 56 b) into and/or aroundwhich the material of the membrane support (10 a) extends.
 3. The wellinsert (10) according to claim 2, wherein said surface features (51, 52,53, 56, 56 a, 56 b) comprise at least one of the following: protrusions(51, 52), recesses (53, 51 a, 51 c), and undercut portions (53).
 4. Thewell insert (10) according to claim 3, wherein said surface features(51, 52, 53, 51 a, 51 c, 56, 56 a, 56 b) comprise a combination of atleast one protrusion (51, 52) and at least one recess (53, 51 a, 51 c),said at least one recess being undercut.
 5. The well insert (10)according to claim 4, wherein each of said protrusion (51, 52) and saidrecess (53, 51 a, 51 c) extend around the periphery of the permeablemembrane (1).
 6. The well insert (10) according to claim 5, wherein saidprotrusion (51, 53) is situated outside of said recess (53, 51 a, 51 c).7. The well insert (10) according to claim 2, wherein said surfacefeatures comprise at least one recess (53) formed as a first groove,said first groove extending around the permeable membrane (1) on aplanar surface thereof, said surface features further comprising aplurality of further grooves (53) arranged perpendicular to said firstgroove.
 8. A method of manufacturing the well insert (10) according toclaim 1, comprising steps of: providing the permeable membrane (1);providing the membrane support (10 a) made of the polyolefin material;placing the membrane support (10 a) in contact with the surface featuresof said permeable membrane (1); melting portions of the membrane support(10 a) in contact with said permeable membrane (1) so as to allow moltenmaterial to flow into the surface features of said permeable membrane(1); and cooling the molten material to fasten said membrane support (10a) to said permeable membrane (1) in the surface features thereof and toseal said membrane support (10 a) to said permeable membrane (1).
 9. Themethod according to claim 8, wherein said step of melting comprisesheating by means of at least one of: laser energy, ultrasonic energy,thermal contact with a hotplate, and infrared radiation.
 10. The methodaccording to claim 8, wherein the surface features (51, 52, 53, 56, 56a, 56 b) of said permeable membrane (1) face said membrane support (10a), said surface features (51, 52, 53, 56, 56 a, 56 b) being arranged tocontact the polyolefin material of the membrane support (10 a), andwherein said step of melting comprises causing the polyolefin materialto flow into contact with said surface features.
 11. The methodaccording to claim 8, wherein said surface features comprise at leastone groove (53), and wherein said step of melting comprises causing saidpolyolefin material to flow into said groove (53).
 12. A well insert(10) for cell culture, comprising: a membrane support (10 a), made ofpolyolefin material and comprising a hanger (70) having an upper end anda lower end, said upper end being adapted to engage a well of amicroplate so as to suspend the well insert (10) therein; and apermeable membrane (1), transparent in both air and water, forsupporting a tissue culture, the permeable membrane (1) being arrangedat said lower end of the hanger (70) and sealed thereto, the permeablemembrane (1) being of brittle material, wherein the membrane support (10a) comprises a seal (65) arranged at said lower end of said hanger (70),and wherein said membrane support (10 a) further comprises an end piece(60) releasably clipped to said hanger (70) so as to support saidpermeable membrane (1) between said end piece (60) and said hanger (70)in contact with said seal, said end piece (60) being arranged so as tocause said permeable membrane (1) to compress said seal upon clipping ofsaid end piece on said hanger.
 13. The well insert (10) according toclaim 12, wherein said end piece (60) is situated outside of said hanger(70).
 14. The well insert (10) according to claim 12, wherein said endpiece (60) comprises a plurality of arms (63) extending towards saidupper end and comprising first clipping element (64) each interfacingwith a corresponding second clipping element (14) provided on saidhanger (70).
 15. The well insert (10) according to claim 14, whereinsaid first clipping element (64) is a hook and said second clippingelement (14) is an opening provided in said hanger.